DOE OSTI · 3364326
Conjugation-based genome engineering enables rapid prototyping and bioproduction in non-model bacteria
Abstract
Abstract Non-model bacteria offer unique metabolic capabilities for sustainable bioproduction, yet their limited genetic accessibility hinders systematic strain development. Here we present conjugation-based serine recombinase-assisted genome engineering (cSAGE), a broad-host-range platform that enables predictable, iterative genomic integration in transformation-resistant bacteria. cSAGE combines conjugative DNA delivery, standardized low-copy vectors, orthogonal recombinases, and modular genetic parts to support rapid pathway assembly and cross-host benchmarking. Using purple nonsulfur bacteria as a testbed, we integrate promoter engineering, multi-payload genome modification, and genome-scale metabolic modeling to empirically evaluate host-dependent pathway performance. Applying this workflow, we identify strain-specific differences in photosynthetic conversion of lignin-derived p -coumarate to the thermoplastic precursor p -vinylphenol. By enabling genome engineering and functional comparison across diverse bacteria using a single plasmid system, cSAGE provides a general framework for non-model strain prototyping and biotransformation discovery.
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Guzman, Michael S. [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0000000238888055), Kiattisewee, Cholpisit [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0000000263517352), Comes, Jackson [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0009000167954162), Robert, Amanda M. [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0009000862506636), Cardiff, Ryan A.L. [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0009000105309010), Cook, Margaret [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0000000342106885), Scott, Allan [Biological Sciences Division, Pacific Northwest National Laboratory Richland, WA 99354, United States] (ORCID:0000000273857633), Alba Burbano, Diego [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0000000284761506), Anastasakis, Stella [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:000900009171396X), Grube, Sarah [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0009000619198303), Heiberg, Kieran [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States] (ORCID:0009000105581938), Darst, Brian [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0009000848281880), Howell, Daniel [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0000000292959774), Dexter, Gara N. [Biosciences Division, Oak Ridge National Laboratory Oak Ridge, TN 37831, United States] (ORCID:0000000216905723), Egbert, Robert G. [Biological Sciences Division, Pacific Northwest National Laboratory Richland, WA 99354, United States] (ORCID:0000000294707124), Zalatan, Jesse G. [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States; Department of Chemistry, University of Washington, Seattle, WA 98195, United States] (ORCID:0000000214580654), Guss, Adam M. [Biosciences Division, Oak Ridge National Laboratory Oak Ridge, TN 37831, United States] (ORCID:0000000158235329), Elmore, Joshua R. [Biological Sciences Division, Pacific Northwest National Laboratory Richland, WA 99354, United States] (ORCID:0000000347509640), Beliaev, Alexander S. [Biological Sciences Division, Pacific Northwest National Laboratory Richland, WA 99354, United States] (ORCID:0000000267664632), Carothers, James M. [Department of Chemical Engineering, University of Washington, Seattle, WA 98195, United States; UW Center for Synthetic Biology and Molecular Engineering & Sciences Institute University of Washington, Seattle, WA 98195, United States] (ORCID:0000000167287833). 2026-04-18. Conjugation-based genome engineering enables rapid prototyping and bioproduction in non-model bacteria. https://doi.org/10.64898/2026.04.17.717921
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